Bacterial type I polyketide synthases (PKSs) are mega-enzyme assembly lines responsible for generating the macrolactone core of a wide range of polyketide products that are biologically active natural compounds (e.g. antimicrobial, antifungal, antiviral, anticancer, and immunosuppressant compounds). Indicative of their significance, polyketide natural products currently form the basis for nearly one-third of pharmaceuticals. PKSs employ a modular multi-step mechanism to produce polyketides, and bioengineering these systems has immense potential for the creation of new chemotypes with invaluable applications in drug discovery. However, such efforts have met with limited success, reflecting our poor structural and mechanistic understanding of the modular process to generate polyketides. We recently employed cryo-electron microscopy (cryo-EM) to show the first subnanometer resolution structures of the full length PikAIII module from the pikromycin PKS biosynthetic pathway, a prototype for assembly-line PKS systems. The findings not only revealed an unexpected module architecture undergoing extensive structural rearrangements, but also showed that the type of substrate linked to a highly mobile acyl carrier protein (ACP) domain specifies its positioning in a way that facilitates assembly-line throughput. By employing recent breakthroughs in cryo-EM technologies, we now aim to obtain high resolution cryo-EM structures of PikAIII and of the terminal PikAIV module, in an effort to resolve several lingering question regarding functional interfaces and module dynamics. Our studies will include both natural and unnatural substrates, seeking to reveal the principles of substrate recognition and processing in these remarkable macromolecular factories. The findings from the proposed studies will for the basis for renewed bioengineering efforts towards the creation of PKSs that can efficiently produce novel compounds of high medicinal value.